Glucose transport in some microorganisms such as, for example, Corynebacterium glutamicum is natively accomplished using the phosphotransferase transport system (PTS). In this system, phosphorylation of glucose is carried out simultaneously to transport. The phospho donor is phosphoenolpyruvate (PEP), therefore linking transport directly to glycolytic flux. In addition, the PTS system is natively regulated by a number of transcriptional processes in ways that are not always ideal for the production of commercial products.
Microbial processes for the production of various commercial products from glucose strive to maximize the efficiency with which the carbon skeleton of glucose is converted into the desired product. Control of glucose flux is critical for the production of products in ways that are dependent on the fermentation process, strain of microbial host being used (e.g., C. glutamicum), and small molecule being produced. If there is too much flux through glycolysis under high concentrations of glucose, glycolytic by-products (usually organic acids) are produced which decrease yield of product. If there is too little transport of glucose into the cell, then it is difficult to produce product at high rates. The genotypes of strains which are engineered in various ways to produce specific products interact with process conditions to lead to situations in which more or less glucose transport occurs than would be ideal to maximize yield or productivity.
Microbial strain improvement has been attempted by the expression of different glucose permeases and glucokinases which may alter glucose transport in such a way as to increase yield or productivity of commercial products. This has been demonstrated in a number of cases. For example, deletion of the native PTS system for glucose transport and overexpression of a native C. glutamicum permease along with a native C. glutamicum kinase led to the increased yield of lysine production from glucose (see Linder et al. Appl. Environ. Microbiol. June 2011 vol. 77, no. 11 pp 3571-3581, the contents of which are hereby incorporated by reference in their entirety). In another example, overexpression of the glucose permease and glucokinase from Z. mobilis in C. glutamicum was used for the production of small molecules (see U.S. Pat. No. 5,602,030, the contents of which are hereby incorporated by reference in its entirety).
However, the selection of a particular glucose permease to create the ideal level of glucose transport for a given metabolic process to produce a specific commercial product relies on a good understanding of a number of interacting factors, including the interaction of the genotype of a strain with the process environment in which fermentation takes place. Further, the correct expression, affinity, and transport rate, in combination with glucose and other carbon source concentrations may be required to deliver a balanced flux of glucose into the cell to match the flux through the pathway of interest. Understanding these parameters a priori and then choosing a single permease which embodies them can be difficult or impossible.
Thus, there is a great need in the art for new methods of engineering industrial microbes for producing specific commercial products, which do not suffer from the aforementioned drawbacks inherent with traditional strain improvement programs.